Pseudo-thermodynamicKinetic Thermodynamic

Introduction

Thermodynamic solubility

Compound Structure LiteratureExperimental

(pH 7.4)

Fenofibrate <1µM <2µM

Ketoconazole 3µM 3µM

DPI 25µM 14µM

Haloperidol 37µM 36µM

Promethazine 55µM 56µM

SOLUBILITY TOOLBOX FOR SUCCESSFUL DESIGN OF

DRUG CANDIDATESFrédéric BELL , Caroline DURAND, Nathalie URBAN

Analytical Sciences Group, Medicinal Chemistry, INVENTIVA 50 rue de Dijon, Daix. 21121- France

Results / Discussion

Protocol :

Strengths :

Protocol : Protocol :

Strengths :Strengths

Sample / compound consumption From stock DMSO solution/1.2 mg

Solubility range 5µM to 100µM

PrincipleDilution to PBS pH 7.4, allow for

precipitation, filtering out and quantitation

Incubation time 30 min

Readout

Reference solution

Ultra Violet(UV)

PBS/ACN (50/50)

Residual solvent 1.3% DMSO

Throughput 20 compounds / 3h

Replicates & quantification4 replicates

1 calibration point (50µm)

RequiresGood UV chromophores

DMSO soluble compounds (10, 5 or 2.5 mM)

Acceptance criteria

Caffeine is our positive control

UV spectrum quality (fig1)

Caffeine : absorbance for reference>0,7AU

Solubility for test solution >110µM

No more than 1 outlier out of mean +/- 15%

Test solutions :min absorbance 0,2AU

No more than 1 outlier out of mean +/- 15%

Sample / compound consumption From stock/ 2mg

Solubility range 5µM to 1mM

PrincipleAddition of PBS pH7.4 into sample

tube, incubation, filtering out and

quantitation

Incubation time 24h

Readout

Reference solution

HPLC-UV or HPLC-MS

50/25/25 (ACN/DMSO/PBS)

Residual solvent none

Throughput 5 compounds / 4h over 2 days

Replicates & quantification3 injections of test solution

4 calibration points

Requires /

Acceptance criteria

Calibration curve :

not less than 3 calibration points

r² >0.975

Test solutions no more than 1

outlier out of mean +/- 15%

Sample / compound consumption From stock DMSO solution/0,2mg

Solubility range 5µM to 200µM

PrincipleEvaporation of DMSO in 96W-plate,

addition of PBS pH7.4, incubation,

centrifugation and quantitation

Incubation time 24h

Readout

Reference solution

Ultra Violet(UV) or HPLC-UV

PBS/DMSO (50/50)

Residual solvent none

Throughput 20 compounds / 4h over 2 days

Replicates & quantification3 replicates for test solutions ,

6 calibration points

RequiresVery good UV chromophores beyond

265nm (DMSO)

DMSO soluble compounds 10mM

Acceptance criteria

caffeine is our the positive control

DPI is used for control chart survey

Calibration curve :

Not less than 4 calibration points

r²> 0.975

test solutions

no more than 1 outlier out of mean+/- 15%

Caffeine solubility should be >=180µM

DPI from 10µM to 30 µM

High throughput

Easy to run

Automation

Visual check of ref solutions

Ability to detect diffusion due to nanoparticles

Sensitive to compound purity. Integrity is checked by LCMS

Sample consumption (~ 1.2mg)

Limited solubility range

Kinetic data, presence of residual DMSO

Restricted to short incubation time

Typical outliers, pitfalls : compound can precipitate in reference

solution; low UV absorbance

Golden reference technique for thermodynamic solubility

Non sensitive to compound purity

Able to check compound integrity over 24 hours

Visual check of reference solutions

Adsorption upon material is circumvented by filtration of

large volumes

Low automation, limited throughput

Compound consumption: neglectable

Thermodynamic-like data (no cosolvent)

Range of interest in Drug Discovery

High throughput in UV readout

Automation

Sensitive to compound purity

Unable to detect degradation over 24 h

HPLC-UV quantitation can be used to check the purity of compounds

Typical outliers, pitfalls : compound remaining stuck to the well

sublimation or degradation of compound during drying (40°C)

These diverse tools are all combined within a unique platform manned by two technical experts. Depending on the stage of the research program, the

optimal combination of data is defined. For early stage programs Kinetic solubility is preferred with indirect measurement of logP whereas for more advanced compounds,

Pseudo-thermodynamic is used as generic. The features of the compounds also influence the selection of the technique (UV-absorbing, expected solubility, sensitivity to

drying…). Finally, for the most advanced compounds, thermodynamic solubility and logD pH of 2.5 and 7.4 remain the gold standard.

Log D is of particular interest when measured at physiological pH of 7.4 . Measurement at low pH for acids or high pH for basic compounds is also worth considering as it corresponds to the LogP value of the neutral

chemical species. To this aim we have developed two HPLC-based indirect techniques, whereas LogD pH of 7.4 is measured by conventional shake-flask technique.

INVENTIVA is a fully integrated Drug Discovery company based in Dijon (France) dedicated to the finding of novel drug development candidates. The chemistry team (>30 Scientists) provides an

extensive panel of services encompassing medicinal chemistry, synthetic & parallel synthesis, Computer-Assisted-Drug-Design, and analytical & purification.

Physical properties play a crucial role in the success of a drug candidate [1]. Compounds with suboptimal physical properties like low solubility not only hamper the reliability of in vitro and in vivo

assays, but also add significant burden to drug development. Even though thermodynamic solubility of lead compounds has always been measured by the Medicinal Chemists, the arrival of

combinatorial chemistry leading to large libraries of screening compounds and small amount of the available samples has generated the need for development of rapid, high throughput, accurate, low

consumption, automated solubility measurement techniques [2]. Typically the concept of kinetic solubility has been introduced [3] to answer this demand, however some limitations of this concept

have been exemplified [4,5] such as the influence of the residual DMSO used for the initial solubilization or the fact that the starting point is not the solid state but the DMSO solution. To overcome

these issues new developments have focused on thermodynamic-like approaches, also called “pseudo-thermodynamic” [6]. Based upon all these techniques, INVENTIVA has set up a full Solubility

Toolbox containing three generic protocols.

Other important parameters in Medicinal Chemistry are partition coefficients (logP/logD). Even if they are systematically predicted using diverse in-silico tools, it is always valuable to benchmark

these predictions against experimental measurements. We therefore added logD measurement option to our toolbox. It is measured via direct or indirect techniques.

As of today, our Physchem team provides on a daily basis the support to our Medicinal Chemistry team.

[1] Lipophilicity and related molecular properties as determinants of pharmacokinetic behavior, B.Testa, P.A.Carrupt, University of Lausanne, CHIMIA, 2000, 54, N°11, 672-677.

[2] A high-throughput screening method for the determination of aqueous drug solubility using Laser nephelometry in microtiter plates, C.D.Bevan, GlaxoWellcome, Analytical Chemistry, Vol 72, N°8, April 15, 2000.

[3] Development of new experimental tools for fast determination of solubility and lipophilicity , Thesis B.Bard, University of Geneva, 2008

[4] Optimizing solubility : kinetic versus thermodynamic solubility temptations and risks, C. Saal, Merck KGaA, European Journal of Pharmaceutical Sciences, 47, 2012, 589-595

[5] A highly automated assay for determining the aqueous equilibrium solubility of Drug Discovery compounds, M.C.Wenlock, AstraZeneca R&D Charnwood, Journal of Laboratory Automation, 2011, 16, 276-284.

[6] Application of dried-DMSO rapid throughput 24h equilibrium solubility in advancing discovery candidates”, Y.W.Alelyunas, AstraZeneca pharmaceuticals LP, European Journal of Pharmaceutical Sciences 37, 2009, 172-182.

Log D

Conclusion

www.inventivapharma.com

Contact : frederic.bell@inventivapharma.com,nathalie.urban@inventivapharma.com

Biomek 3000 liquid handlerExample of evaporated plate

Weaknesses :

AbbreviationsACN : acetonitrile, DMSO : dimethylsulfoxyde, PBS : Phosphate Buffer Solution,

DPI : 4,5-diphenylimidazole

Weaknesses :

Weaknesses :

Fig 3 : Structure-Property-Relationship established over 6 closely related compounds by Kinetic protocol

Fig 2 : Control charts for kinetic solubility protocol highlighting the very

good reproducibility of the technique (caffeine)

The kinetic protocol is basically a high-throughput protocol which

is very stable (fig2) and robust. Data quality remains excellent

as illustrated in fig3. This example illustrates that tiny structural

differences can be easily discriminated.

Whereas determination of aqueous solubility by thermodynamic approach is not suitable for high throughput,

particularly when the amount of compounds is limited or when the compounds are prepared by parallel

synthesis, Kinetic protocol offers a first option easy to set up and to automate. As an alternative, the Pseudo-

thermodynamic protocol, as introduced by Y.W.Alelyunas et al, is able to deliver a very good compromise

between compound consumption, throughput and data quality (thermodynamic-like data).

Fig 4 : control charts for Pseudo-thermodynamic protocol exhibiting the

good reproducibility of the technique.

However this protocol requires a high level of

expertise as several steps are critical and

must be carefully developed :

- DMSO evaporation,

- stirring of plates,

- withdrawing of centrifugated solutions

Lim

ited

a

mo

un

ts in

D

MS

O s

olu

tion

Hig

h

Th

rou

gh

pu

t

Compound

ID

Chemical

StructureMW Formula

Kinetic

Solubility

Compound

ID

Chemical

StructureMW Formula

Kinetic

Solubility

IV1605435-01 338C22 H30

N2 O55µM IV1608474-01 326

C20 H26

N2 O2>100µM

IV1605440-01 340C21 H28

N2 O274µM IV1608473-01 350

C22 H26

N2 O273µM

IV1605431-01 352C22 H28

N2 O290µM IV1608475-01 363

C22 H25

N3 O297µM

All the protocols can be performed at different pH and in various media.

The required equipment is limited to a liquid handling platform equipped with a UV-plate reader

and completed by one HPLC-UV-MS instrument.

Fig 1 : UV spectrum is inspected

for absence of diffusion

Table 1: validation set (literature is retained if conditions are detailed)

0

0,1

0,2

0,3

0,4

0,5

0,6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Op

tica

l d

ensi

ty (

Au)

Runs

Control chart- Reference compound

Caffeine (Test plate)

0

0,2

0,4

0,6

0,8

1

1,2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Op

tica

l d

ensi

ty (

Au)

Runs

Control chart - Reference compound

Caffeine (130µM Reference plate)

0

5

10

15

20

25

30

35

40

1 2 3 4 5 6 7 8 9 10 11 12

So

lub

ilit

y (

µM

)

Runs

Controle chart - Reference compound

DPI (Test plate)

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1 2 3 4 5 6 7 8 9 10 11

Op

tica

l d

ensi

ty (

Au)

Runs

Control chart- Reference control

Caffeine (Test plate)